CN107267492A - A kind of expression of Halase recombinant protein - Google Patents
A kind of expression of Halase recombinant protein Download PDFInfo
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- CN107267492A CN107267492A CN201710556935.1A CN201710556935A CN107267492A CN 107267492 A CN107267492 A CN 107267492A CN 201710556935 A CN201710556935 A CN 201710556935A CN 107267492 A CN107267492 A CN 107267492A
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- expression
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- halase
- recombinant plasmid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6418—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals from snakes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The present invention relates to a kind of expression of Halase recombinant protein.This method comprises the following steps：(1) the Halase gene after optimization Halase gene (2) PCR amplifications optimization；(3) Agkis pMCX expression vectors are built, plasmid is converted in competent escherichia coli cell and expanded, goes out positive colony through Amp resistance screenings, is sequenced, the correct recombinant plasmid of sequencing is extracted；(4) by Transfected Recombinant Plasmid to Chinese hamster ovary celI；(5) expression of recombinant proteins is identified.The present invention is expressing cho cell one kind restructuring Halase first using serum free suspension formula culture, with bioengineering means, the actual production problem unstable compared with snake venom products material source deficiency, quality is successfully solved.
Description
Technical field
The present invention relates to a kind of expression of Halase recombinant protein.
Background technology
Halase (Haemocoagulase Acutus, be abbreviated as Halase) is a class of this team research and development
Stop blooding new drug, is a kind of batroxobin isolated and purified from agkistrodon acutus (Agkistrodonacutus) snake venom, with stronger
Anastalsis, and safety non-toxic.
At present, the source of poisonous snake is mainly that artificial feeding and field catch two ways, different geographical environment and raising
The factors such as condition so that the composition and property of snake venom all have very big difference, are that large-scale production causes certain difficulty,
The homogeneity of product is restricted.With the development of biotechnology, exploitation snake venom thrombin-like enzyme (Snake Venom
Thrombin-Like Enzymes, SVTLEs) recombinant protein product substitutes this kind of natural products becomes SVTLEs medicament research and developments
An important directions, to pass through the means of protein engineering, stable SVTLEs product of the expression with good physiologically active.
For the structure of SVTLEs expression of recombinant proteins systems, carried out earliest by prokaryotic expression system.Although
SVTLEs glycosylation modified situation is not quite similar, but major part SVTLEs is glycoprotein, and content is individual between 0%-30%
Other species is even as high as more than 40%.And conventional bacterial expression strain does not have glycosylated ability, this will be largely effected on
The activity of albumen.And the expression of Escherichia coli exists with inclusion bodies more, researcher usually requires to add sulphur hydrogen reduction
Albumen (thioredoxin, TrxR) label increases the solubility expression of foreign protein.It is more ripe in eukaryotic expression system
Yeast expression system used also as SVTLEs expression of recombinant proteins.But yeast expression system is for the expression of foreign protein
It there may be excessive glycosylation modified, or lack the modification of complexity sugar chain, these can also influence the function of recombinant protein.
Mammalian cell expression system has been increasingly becoming heterologous glycoprotein protein expression because of the glycosylation machinery with higher level
Important system.In mammalian cell expression system, Chinese hamster ovary celI has become the most important expression of field of biological pharmacy
System, the development of serum free suspension culture technique greatly improve traditional mammalian cell expression system susceptible viral infection,
The low limitation of automatization level, is more suitable for industrialization large-scale production.So its application is constantly expanded, extensively should
Research and development for biological medical products such as antibody, vaccine, recombinant protein medicines.Food and medicine Surveillance Authority of the U.S. was in 2010
CHO expressing proteins are have approved directly to be used as medicine.
The content of the invention
To solve above-mentioned technical problem：The application proposes a kind of expression of Halase recombinant protein, first
Secondary utilization Chinese hamster ovary celI expresses a kind of Halase recombinant protein, including following step by serum free suspension culture technique
Suddenly：
(1) Halase gene is optimized；
(2) the Halase gene of PCR amplifications optimization；
(3) construction of expression vector；
(4) by Transfected Recombinant Plasmid to Chinese hamster ovary celI；
(5) expression of recombinant proteins is identified.
Wherein, the construction method of step (3) expression vector is specially：By the PCR primer of recovery and pMCX cloning vector enzymes
Connection is cut, Agkis-pMCX plasmids are built, Agkis-pMCX plasmids are converted in competent escherichia coli cell and expanded, through Amp
Resistance screening goes out positive colony, is sequenced；Extract the correct recombinant plasmid of sequencing.
The PCR primer is with pMCX cloning vector digestion connection method method for optimizing：Agkistrodon acutus after amplification are coagulated
Hemase gene carries out endonuclease digestion, and gene purpose fragment is connected on expression vector using T4 ligases.
The specific method of Transfected Recombinant Plasmid to Chinese hamster ovary celI is by step (4)：Take cell to centrifuge in right amount, use fresh solution
It is resuspended, it is 5mioc/mL to make final cell densities；During centrifugation, DNA and PEI are premixed, incubation at room temperature；The DNA＆ handled well
PEI mixtures, are added in the cell of preparation, mix, 31 DEG C of cultures；Transfection, adds appropriate DMSO, rocks mixing, 31 DEG C
Culture；Sample is taken to be detected after transfection；Wherein the recombinant plasmid comprising protein gene total length is individually transfected, the base of subunit containing A
Because the recombinant plasmid cotransfection of sequence and B subunit gene sequences is to Chinese hamster ovary celI.
The specific method of the step (1) comprises the following steps：
1) Halase gene order is optimized according to the encoding preferences of mammal；
2) designed according to the Halase gene of synthesis in sense primer and anti-sense primer, primer and introduce Not I
With I two restriction enzyme sites of BamH, one section of secretory signal peptide sequence is also added in addition；
3) gene contains two subunit sequences of A subunits and B subunits of protein, passes through the tune to upstream and downstream primer
It is whole, protein gene sequence, A subunit genes sequence and B subunit gene sequences can be amplified respectively.
Relative to prior art, the present invention is expressing cho cell one kind restructuring first using serum free suspension formula culture
Halase, with bioengineering means, compared with successfully solving, snake venom products material source deficiency, quality are unstable
Actual production problem.
Brief description of the drawings
Fig. 1 is the schematic diagram of optimum combination Halase gene order of the embodiment of the present invention.
Fig. 2 is that the embodiment of the present invention recombinates Halase SDS-PAGE electrophoresis schematic diagrames.
Fig. 3 is the schematic diagram that the embodiment of the present invention recombinates Halase Western Blot
Embodiment
With reference to specific embodiment, the present invention is described further.
Embodiment：The operating method of Halase expression of recombinant proteins
1. experiment material
1.1 laboratory apparatus
PCR instrument：Bio-Rad companies；Electrophoresis apparatus：Beijing Jun Yi east electrophoresis equipment Co., Ltd；Gel imaging system：Pearl
Extra large unexpected rival Instruments Medicaux G.B. Inc.；Bale cutting instrument：Hangzhou meter Ou Instrument Ltd.；Constant temperature blending instrument：Rice Europe, Hangzhou instrument has
Limit company；Ultramicron ultraviolet-uisible spectrophotometer：Quawell companies of the U.S. 0；2-8 degree refrigerating boxes：The U.S. water chestnut low temperature science and technology of middle section
Co., Ltd；- 20 degree medical low temperature boxes：The U.S. water chestnut low temperature science and technology limited Company of middle section；Three hole electric heating constant temperature tanks：On
Hai Hengyi scientific instrument Co., Ltd；Bacteriological incubator：Shanghai one scientific instrument Co., Ltd of perseverance；Horizontal full temperature shaken cultivation
Case：Shanghai Zhi Chu Instrument Ltd.；Cell culture table：Adolf Kuhner companies；Ultrasonic cell disruptor：Ningbo is new
Sesame biotech inc；Magnetic stirring apparatus：Wiggens companies；PH meter：Ohaus Instrument (Shanghai) Co., Ltd.；
Double clean work station：Shanghai Su Jing Industrial Co., Ltd.s；Transferring system：Bio-Rad companies；Vertical electrophoresis system：Bio-Rad
Company；Decolorization swinging table：Shanghai Tian Neng Science and Technology Ltd.s；High-pressure sterilizing pot：Shenan Medical Appliances Factory, Shanghai.
1.2 experiment reagent
Expression vector pMCX, bacillus coli DH 5 alpha, CHO DG44 cells rise bio tech ltd's system by Guang Zhouhan
Standby, offer.KOD archaeal dna polymerases：(Shanghai) bio tech ltd is spun by Japan；T4DNA ligases：Offshore protein science and technology
Co., Ltd；Restriction enzyme Not I：Thermo Fisher Scientific companies；Restriction enzyme BamH I：
Thermo Fisher Scientific companies；Plasmid extraction kit：OMEGA companies of the U.S.；DNA Ago-Gels reclaim examination
Agent box：OMEGA companies of the U.S.；DNA Marker：Thermo Fisher Scientific companies；CHO nutrient solutions：Lonza is public
Department；DMSO：Tianjin Chemical Reagents Factory No.1；PEI：Sigma aldrich companies；Trypan blue dye liquor：Sigma aldrich are public
Department；Specific polyclonal antibody：Cloud-clone companies；Rabbit IgG antibody：Proteintech companies；Ampicillin：It is raw
Work bio tech ltd；Agar powder：Guangdong Huan Kai bio tech ltd；Yeast extract：Oxoid companies；Peptone：
Oxoid companies.
2. experimental method
2.1 gene magnification
The protein sequence of Halase is translated by JCAT (Java Codon Adaptation Tool)
Sequence is optimized into DNA sequence dna, and according to the encoding preferences of mammal, the gene order See Figure 1 after optimization, should
Gene order is synthesized by Guangzhou Ai Ji Bioisystech Co., Ltd.
Designed according to the Halase gene of synthesis in sense primer and anti-sense primer, primer and introduce the Hes of Not I
I two restriction enzyme sites of BamH, also added one section of secretory signal peptide sequence, are shown in Table 1 in addition.
Table 1 recombinates the PCR primer of Halase
The gene order contains two subunits (A and B) sequence of protein, can by the adjustment to upstream and downstream primer
Protein gene sequence, A subunit genes sequence and B subunit gene sequences are amplified respectively.Enter performing PCR expansion to synthetic gene
Increase, PCR primer is reclaimed with agarose gel electrophoresis.
Endonuclease digestion is carried out to the Halase gene after amplification, using T4 ligases gene purpose fragment
It is connected on expression vector.Screening antibiotic is used as from ampicillin (Ampicillin, Amp).
2.2 expression vector establishment
The PCR primer of recovery is connected with pMCX cloning vector digestions, Agkis-pMCX plasmids are built.By Agkis-pMCX
Expanded in plasmid conversion competent escherichia coli cell, go out positive colony through Amp resistance screenings, send Guangzhou Ai Ji biotechnologys to have
Limit company is sequenced.The correct recombinant plasmid of sequencing is extracted, as follow-up Chinese hamster ovary celI transfection assay.
2.3CHO cell transfecting
The transfection same day, record cell density (mioc/mL) takes cell to centrifuge in right amount, 1300rpm, 3min；Use fresh solution
(31 DEG C, preheat in advance) are resuspended, and it is 5mioc/mL to make final cell densities.During centrifugation, by DNA and PEI in clean centrifugation
Premix, gently blow and beat in pipe, be incubated at room temperature 3-5min.The DNA＆PEI mixtures handled well, are added in the cell of preparation, shake
Shake and mix, 31 DEG C of cultures.Turn and then 10min, add appropriate DMSO, rock mixing, 31 DEG C of cultures.Sample is taken to carry out after transfection
Detection.Wherein the recombinant plasmid comprising protein gene total length is individually transfected, the sequence of subunit gene containing A and B subunit gene sequences
Recombinant plasmid cotransfection to Chinese hamster ovary celI.
2.4 protein expressions are identified
(1) sample preparation
Take after cell 80ul, the 1300rpm centrifugation 3min for having cultivated a couple of days, supernatant cell precipitation is collected respectively, is drawn
5 × loading Buffer that upper honest and upright and thrifty 80ul adds 20ul are mixed, and cell adds 1 × PBS of 80ul (pH7.5) buffer solution
It is resuspended after cell and adds after 20 μ l (reduced) 5 × loading Buffer mixings, 100 DEG C is boiled sample 8min, 12000rpm centrifugation
1min is stand-by/it is stored in -20 DEG C of refrigerators.
(2)SDS-PAGE
First piece of glue of electrophoresis：The polyacrylamide gel of 12% concentration of configured in advance, per the μ l of hole loading 15, with 80V electricity
Pressure runs to go out to concentrate to sample after glue is adjusted to 120V rear electrophoresis about 90min by voltage.Second piece of glue：12% concentration of configured in advance
Polyacrylamide gel, per the μ l of hole loading 15, is run to sample to go out to concentrate after glue with 80V voltages voltage is adjusted into 120V rear electrophoresis about
90min.As shown in Fig. 2 wherein, M is albumen Marker；Cell is clasmatosis liquid；Sup is cell culture supernatant.
(3)Western blot
80mA transferring film 40min, add 5% skimmed milk power room temperature closing 1h.Specific antibody is according to 1：5000 ratio room
Temperature is incubated 2h；TBST is washed 3 times, each 5min；Rabbit source anti-igg antibody incubation at room temperature 1h；PBST is washed 3 times, each 5min；
ECL develops the color, and exposure 1min takes pictures.As shown in figure 3, wherein, M is albumen Marker；Cell is clasmatosis liquid；Sup is cell
Culture supernatant.
3 experimental results
SDS-PAGE is shown, is carried containing this six kinds restructuring of HGFs, Heavy, Acti, Light, HA+HB, LA+HB
Detection has the protein close with target protein molecular weight in the clasmatosis liquid of body, and then intimate in cell culture supernatant
There is no bands visible.The further Testing and appraisals of Western Blot are shown, in addition to HGFs, Heavy, Acti, Light,
Detection has the expression of target protein in the clasmatosis liquid of this five kinds of recombinant vectors of HA+HB, LA+HB, and molecular weight is left in 32kDa
It is right；And there is no bands visible in cell culture supernatant.
Claims (5)
1. a kind of expression of Halase recombinant protein, successively including Halase gene optimization, excellent
Change gene PCR amplification, expression vector establishment, Transfected Recombinant Plasmid and expression of recombinant proteins authentication step, it is characterised in that：It is described
Expression vector establishment method for the PCR primer of recovery is connected with pMCX cloning vector digestions, structure Agkis-pMCX plasmids,
Agkis-pMCX plasmids are converted in competent escherichia coli cell and expanded, goes out positive colony through Amp resistance screenings, is surveyed
Sequence；Extract the correct recombinant plasmid of sequencing；Described Transfected Recombinant Plasmid method be by Transfected Recombinant Plasmid to Chinese hamster ovary celI, its
In individually transfected comprising the recombinant plasmid of protein gene total length, the restructuring matter of the sequence of subunit gene containing A and B subunit gene sequences
Grain cotransfection is to Chinese hamster ovary celI.
2. expression as claimed in claim 1, it is characterised in that：Described Transfected Recombinant Plasmid method is specially：Take thin
Born of the same parents centrifuge in right amount, are resuspended with fresh solution, and it is 5mioc/mL to make final cell densities；During centrifugation, DNA and PEI are premixed, room
Temperature is incubated 3-5min；The DNA＆PEI mixtures handled well, are added in the cell of preparation, mix, 31 DEG C of cultures；After transfection
10min, adds appropriate DMSO, rocks mixing, 31 DEG C of cultures；Sample is taken to be detected after transfection；Wherein comprising protein-based
Because the recombinant plasmid of total length is individually transfected, the recombinant plasmid cotransfection of the sequence of subunit gene containing A and B subunit gene sequences to CHO
Cell.
3. expression as claimed in claim 1 or 2, it is characterised in that：It will be reclaimed in described expression vector establishment method
PCR primer be with the specific method that pMCX cloning vector digestions are connected：Endonuclease digestion is carried out to PCR primer, connected using T4
Enzyme is connect to be connected on expression vector gene purpose fragment.
4. expression as claimed in claim 1, it is characterised in that：Described Halase genetic optimization method bag
Include following steps：
(1) Halase gene order is optimized according to the encoding preferences of mammal；
(2) designed according to the Halase gene of synthesis in sense primer and anti-sense primer, primer and introduce the Hes of Not I
I two restriction enzyme sites of BamH, also added one section of secretory signal peptide sequence in addition；
(3) gene contains two subunit sequences of A subunits and B subunits of protein, can by the adjustment to upstream and downstream primer
Protein gene sequence, A subunit genes sequence and B subunit gene sequences are amplified respectively.
5. expression as claimed in claim 1, it is characterised in that：The expression of recombinant proteins authentication method includes following step
Suddenly：
(1)SDS-PAGE：First piece of glue of electrophoresis：The polyacrylamide gel of 12% concentration of configured in advance, per the μ l of hole loading 15,
Run to sample to go out to concentrate after glue with 80V voltages and voltage is adjusted to 120V rear electrophoresis about 90min；Second piece of glue：Configured in advance
The polyacrylamide gel of 12% concentration, per the μ l of hole loading 15, is run to sample to go out to concentrate after glue with 80V voltages and is adjusted to voltage
120V rear electrophoresis about 90min；
(2)Western blot：80mA transferring film 40min, add 5% skimmed milk power room temperature closing 1h；Specific antibody is according to 1：
5000 ratio incubation at room temperature 2h；TBST is washed 3 times, each 5min；Rabbit source anti-igg antibody incubation at room temperature 1h；PBST washings 3
It is secondary, each 5min；ECL develops the color, and exposure 1min takes pictures.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986813A (en) * | 2006-12-18 | 2007-06-27 | 中国人民解放军军事医学科学院生物工程研究所 | Batroxobin and its preparing process and specific coding gene |
| CN101688195A (en) * | 2007-07-06 | 2010-03-31 | 阿斯利康(瑞典)有限公司 | Method for production of recombinant human thrombin `644 |
| CN102660565A (en) * | 2012-04-25 | 2012-09-12 | 郑颖 | Agkistrodon acutus hemocoagulase gene and methods for preparing expression vector, host cell and recombinant protein thereof |
-
2017
- 2017-07-10 CN CN201710556935.1A patent/CN107267492A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986813A (en) * | 2006-12-18 | 2007-06-27 | 中国人民解放军军事医学科学院生物工程研究所 | Batroxobin and its preparing process and specific coding gene |
| CN101688195A (en) * | 2007-07-06 | 2010-03-31 | 阿斯利康(瑞典)有限公司 | Method for production of recombinant human thrombin `644 |
| CN102660565A (en) * | 2012-04-25 | 2012-09-12 | 郑颖 | Agkistrodon acutus hemocoagulase gene and methods for preparing expression vector, host cell and recombinant protein thereof |
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Application publication date: 20171020 |